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On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.

Bachmann A, Draga M, Grawe F, Knust E - BMC Dev. Biol. (2008)

Bottom Line: Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains.Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. bachmana@uni-duesseldorf.de

ABSTRACT

Background: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.

Results: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.

Conclusion: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

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varicose encodes a MAGUK protein. (A) Exon-intron structure of the vari locus (scale bar according to FlyBase). Two different transcripts could be isolated: the vari-long transcript contains exons 1, 2, 3 to 10 with the translational start residing in exon 1; vari-short consists of exons 3a, 3 to 10 with the translational start site in exon 4 (dark grey boxes: exons common to both transcripts, light grey boxes: exons specific to vari-long, white boxes: UTR). Mobilisation of the P(XP)-element d10880 yielded the vari-short-specific mutant allele variMD109, which removes exon 3a and adjacent 3'-intronic DNA (shaded box). Primer pairs vari-long-5/3 and vari-short-5/3 were used to detect the corresponding vari transcripts in the wild-type (D) and different vari alleles (see Fig. 5). (B) Structure and size comparison of the MAGUK proteins Vari-long and Vari-short with their human homologs hsMPP6_c (GenBank accession number EAW93810), hsMPP6_a (GenBank accession number EAW93808) and hsMPP2_b (GenBank accession number EAW51656), respectively. The percentages of amino acid identities of the domains with respect to the corresponding domains of Vari-long are shown. (C) Northern blot of 5 μg poly(A+) RNA from staged wild-type embryos (1 = 0–4 h, 2 = 4–12 h, 3 = 12–24 h) hybridised with a probe that detects both vari-long and vari-short transcripts. (D) RT-PCR on total RNA from wild-type embryos (> 8 h old) with primer pairs vari-long-5/3 and vari-short-5/3 detects single vari-long- and vari-short-transcripts. (E) Western blot analysis of protein lysates from staged wild-type embryos and embryos of different genotypes (> 8 h old), probed with an anti-Vari antibody that recognises both Vari-long and Vari-short (1 = wt, 0–4 h, 2 = wt, 4–12 h, 3 = wt, 12–24 h, 4 = daG32>UAS Flag-vari-long, 5 = daG32>UAS vari-RNAi, 6 = Df(2L)DS6). Overexpressed Flag-Vari-long in lane 4 is slightly bigger than endogenous Vari-long due to the N-terminal Flag-tag. The protein amount loaded per lane equals 5 embryos with the exception of lysates from daG32>UAS Flag-vari-long embryos (equals 0.5 embryos). The lysate from Df(2L)DS6 embryos serves as a negative control and an unspecific, cross-reacting band (asterisk) as a loading control.
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Figure 1: varicose encodes a MAGUK protein. (A) Exon-intron structure of the vari locus (scale bar according to FlyBase). Two different transcripts could be isolated: the vari-long transcript contains exons 1, 2, 3 to 10 with the translational start residing in exon 1; vari-short consists of exons 3a, 3 to 10 with the translational start site in exon 4 (dark grey boxes: exons common to both transcripts, light grey boxes: exons specific to vari-long, white boxes: UTR). Mobilisation of the P(XP)-element d10880 yielded the vari-short-specific mutant allele variMD109, which removes exon 3a and adjacent 3'-intronic DNA (shaded box). Primer pairs vari-long-5/3 and vari-short-5/3 were used to detect the corresponding vari transcripts in the wild-type (D) and different vari alleles (see Fig. 5). (B) Structure and size comparison of the MAGUK proteins Vari-long and Vari-short with their human homologs hsMPP6_c (GenBank accession number EAW93810), hsMPP6_a (GenBank accession number EAW93808) and hsMPP2_b (GenBank accession number EAW51656), respectively. The percentages of amino acid identities of the domains with respect to the corresponding domains of Vari-long are shown. (C) Northern blot of 5 μg poly(A+) RNA from staged wild-type embryos (1 = 0–4 h, 2 = 4–12 h, 3 = 12–24 h) hybridised with a probe that detects both vari-long and vari-short transcripts. (D) RT-PCR on total RNA from wild-type embryos (> 8 h old) with primer pairs vari-long-5/3 and vari-short-5/3 detects single vari-long- and vari-short-transcripts. (E) Western blot analysis of protein lysates from staged wild-type embryos and embryos of different genotypes (> 8 h old), probed with an anti-Vari antibody that recognises both Vari-long and Vari-short (1 = wt, 0–4 h, 2 = wt, 4–12 h, 3 = wt, 12–24 h, 4 = daG32>UAS Flag-vari-long, 5 = daG32>UAS vari-RNAi, 6 = Df(2L)DS6). Overexpressed Flag-Vari-long in lane 4 is slightly bigger than endogenous Vari-long due to the N-terminal Flag-tag. The protein amount loaded per lane equals 5 embryos with the exception of lysates from daG32>UAS Flag-vari-long embryos (equals 0.5 embryos). The lysate from Df(2L)DS6 embryos serves as a negative control and an unspecific, cross-reacting band (asterisk) as a loading control.

Mentions: In Drosophila epithelia, the MAGUK-proteins Stardust (Sdt) and Discs Large (Dlg) are required to organise protein complexes in the subapical and the lateral membrane, respectively. Sdt recruits the transmembrane protein Crumbs (Crb) and two other scaffolding proteins, DLin-7 and DPATJ, into a complex, which is localised subapically, i. e. in a small region of the apical plasma membrane just apical to the zonula adherens, in epithelial and photoreceptor cells [reviewed in [23]]. We were interested in identifying additional partners of the Crumbs complex, which might have a function in the organisation of epithelia of the Drosophila embryo. Therefore, we took advantage of the recently published protein interaction map [24] to screen for partners of DLin-7, a component of the Crumbs complex [5,25]. One of the putative partners identified in this screen is encoded by Drosophila CG9326, located at 38E10 on the left arm of the second chromosome. It is predicted to encode three isoforms, which differ in their size and are the result of differential transcriptional initiation and alternative splicing (FlyBase) (Fig. 1A). As demonstrated recently [13], these proteins are encoded by varicose (vari; see below) and define a new subgroup of MAGUK proteins, which includes the mammalian proteins MPP2 and MPP6/VAM-1/Pals2 (Fig. 1B). Previously, only a single L27 domain was predicted in the longer isoform. When using SMART and reciprocal BLASTs (see Materials and Methods), two L27 domains are predicted. This is in agreement with the domain structure of orthologues from the honeybee, mouse, Xenopus and zebrafish (see Additional file 1). Strikingly, human MPP6 also encodes two isoforms of different size, MPP6_a and MPP6_c, the longer of which carries an extended N-terminal region with two L27 domains.


On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.

Bachmann A, Draga M, Grawe F, Knust E - BMC Dev. Biol. (2008)

varicose encodes a MAGUK protein. (A) Exon-intron structure of the vari locus (scale bar according to FlyBase). Two different transcripts could be isolated: the vari-long transcript contains exons 1, 2, 3 to 10 with the translational start residing in exon 1; vari-short consists of exons 3a, 3 to 10 with the translational start site in exon 4 (dark grey boxes: exons common to both transcripts, light grey boxes: exons specific to vari-long, white boxes: UTR). Mobilisation of the P(XP)-element d10880 yielded the vari-short-specific mutant allele variMD109, which removes exon 3a and adjacent 3'-intronic DNA (shaded box). Primer pairs vari-long-5/3 and vari-short-5/3 were used to detect the corresponding vari transcripts in the wild-type (D) and different vari alleles (see Fig. 5). (B) Structure and size comparison of the MAGUK proteins Vari-long and Vari-short with their human homologs hsMPP6_c (GenBank accession number EAW93810), hsMPP6_a (GenBank accession number EAW93808) and hsMPP2_b (GenBank accession number EAW51656), respectively. The percentages of amino acid identities of the domains with respect to the corresponding domains of Vari-long are shown. (C) Northern blot of 5 μg poly(A+) RNA from staged wild-type embryos (1 = 0–4 h, 2 = 4–12 h, 3 = 12–24 h) hybridised with a probe that detects both vari-long and vari-short transcripts. (D) RT-PCR on total RNA from wild-type embryos (> 8 h old) with primer pairs vari-long-5/3 and vari-short-5/3 detects single vari-long- and vari-short-transcripts. (E) Western blot analysis of protein lysates from staged wild-type embryos and embryos of different genotypes (> 8 h old), probed with an anti-Vari antibody that recognises both Vari-long and Vari-short (1 = wt, 0–4 h, 2 = wt, 4–12 h, 3 = wt, 12–24 h, 4 = daG32>UAS Flag-vari-long, 5 = daG32>UAS vari-RNAi, 6 = Df(2L)DS6). Overexpressed Flag-Vari-long in lane 4 is slightly bigger than endogenous Vari-long due to the N-terminal Flag-tag. The protein amount loaded per lane equals 5 embryos with the exception of lysates from daG32>UAS Flag-vari-long embryos (equals 0.5 embryos). The lysate from Df(2L)DS6 embryos serves as a negative control and an unspecific, cross-reacting band (asterisk) as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: varicose encodes a MAGUK protein. (A) Exon-intron structure of the vari locus (scale bar according to FlyBase). Two different transcripts could be isolated: the vari-long transcript contains exons 1, 2, 3 to 10 with the translational start residing in exon 1; vari-short consists of exons 3a, 3 to 10 with the translational start site in exon 4 (dark grey boxes: exons common to both transcripts, light grey boxes: exons specific to vari-long, white boxes: UTR). Mobilisation of the P(XP)-element d10880 yielded the vari-short-specific mutant allele variMD109, which removes exon 3a and adjacent 3'-intronic DNA (shaded box). Primer pairs vari-long-5/3 and vari-short-5/3 were used to detect the corresponding vari transcripts in the wild-type (D) and different vari alleles (see Fig. 5). (B) Structure and size comparison of the MAGUK proteins Vari-long and Vari-short with their human homologs hsMPP6_c (GenBank accession number EAW93810), hsMPP6_a (GenBank accession number EAW93808) and hsMPP2_b (GenBank accession number EAW51656), respectively. The percentages of amino acid identities of the domains with respect to the corresponding domains of Vari-long are shown. (C) Northern blot of 5 μg poly(A+) RNA from staged wild-type embryos (1 = 0–4 h, 2 = 4–12 h, 3 = 12–24 h) hybridised with a probe that detects both vari-long and vari-short transcripts. (D) RT-PCR on total RNA from wild-type embryos (> 8 h old) with primer pairs vari-long-5/3 and vari-short-5/3 detects single vari-long- and vari-short-transcripts. (E) Western blot analysis of protein lysates from staged wild-type embryos and embryos of different genotypes (> 8 h old), probed with an anti-Vari antibody that recognises both Vari-long and Vari-short (1 = wt, 0–4 h, 2 = wt, 4–12 h, 3 = wt, 12–24 h, 4 = daG32>UAS Flag-vari-long, 5 = daG32>UAS vari-RNAi, 6 = Df(2L)DS6). Overexpressed Flag-Vari-long in lane 4 is slightly bigger than endogenous Vari-long due to the N-terminal Flag-tag. The protein amount loaded per lane equals 5 embryos with the exception of lysates from daG32>UAS Flag-vari-long embryos (equals 0.5 embryos). The lysate from Df(2L)DS6 embryos serves as a negative control and an unspecific, cross-reacting band (asterisk) as a loading control.
Mentions: In Drosophila epithelia, the MAGUK-proteins Stardust (Sdt) and Discs Large (Dlg) are required to organise protein complexes in the subapical and the lateral membrane, respectively. Sdt recruits the transmembrane protein Crumbs (Crb) and two other scaffolding proteins, DLin-7 and DPATJ, into a complex, which is localised subapically, i. e. in a small region of the apical plasma membrane just apical to the zonula adherens, in epithelial and photoreceptor cells [reviewed in [23]]. We were interested in identifying additional partners of the Crumbs complex, which might have a function in the organisation of epithelia of the Drosophila embryo. Therefore, we took advantage of the recently published protein interaction map [24] to screen for partners of DLin-7, a component of the Crumbs complex [5,25]. One of the putative partners identified in this screen is encoded by Drosophila CG9326, located at 38E10 on the left arm of the second chromosome. It is predicted to encode three isoforms, which differ in their size and are the result of differential transcriptional initiation and alternative splicing (FlyBase) (Fig. 1A). As demonstrated recently [13], these proteins are encoded by varicose (vari; see below) and define a new subgroup of MAGUK proteins, which includes the mammalian proteins MPP2 and MPP6/VAM-1/Pals2 (Fig. 1B). Previously, only a single L27 domain was predicted in the longer isoform. When using SMART and reciprocal BLASTs (see Materials and Methods), two L27 domains are predicted. This is in agreement with the domain structure of orthologues from the honeybee, mouse, Xenopus and zebrafish (see Additional file 1). Strikingly, human MPP6 also encodes two isoforms of different size, MPP6_a and MPP6_c, the longer of which carries an extended N-terminal region with two L27 domains.

Bottom Line: Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains.Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. bachmana@uni-duesseldorf.de

ABSTRACT

Background: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.

Results: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.

Conclusion: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

Show MeSH
Related in: MedlinePlus